Inositol phospholipids have emerged as important key players in a wide variety of cellular functions. Among the seven existing inositol phospholipids, phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P(2)) has attracted much attention in recent years due to its important role in numerous cellular signaling events and regulations, which in turn impact several human diseases. This particular lipid is recognized in the cell by specific lipid binding domains, such as the Pleckstrin-homology (PH) domain, which is also employed as a tool to monitor this important lipid. Here, we describe the synthesis and biological characterization of a small molecule that mimics the PH domain as judged by its ability to bind specifically to only PI(4,5)P(2) and effectively compete with the PH domain in vitro and in a cellular environment. The binding constant of this small molecule PH domain mimetic (PHDM) was determined to be 17.6 ± 10.1 μM, similar in potency to the PH domain. Using NIH 3T3 mouse fibroblast cells we demonstrated that this compound is cell-permeable and able to modulate PI(4,5)P(2)-dependent effects in a cellular environment such as the endocytosis of the transferrin receptor, loss of mitochondria, as well as stress fiber formation. This highly PI(4,5)P(2)-specific chemical mimetic of a PH domain not only is a powerful research tool but might also be a lead compound in future drug developments targeting PI(4,5)P(2)-dependent diseases such as Lowe syndrome.